The present invention relates to novel analogs of fusidic acid. In particular, the present invention relates to novel glycosylated analogs of fusidic acid. The present invention also relates to novel glycosylated analogs of fusidic acid that have different solubility properties than unmodified fusidic acid and that act as chemotherapeutic agents.
The discovery of antibiotics was possibly the most important medical breakthrough of the twentieth century, making many previously lethal microbial infections easily treatable. However, the benefits of antibiotic therapy have gradually given rise to a dangerous development, namely antibiotic-resistant microorganisms. Through the constant use, and often overuse, of antibiotics, mankind has begun the process of selecting strains of bacteria which are resistant to many types of antibiotics.
Physicians employ many strategies to deal with antibiotic resistance, including: aggressively searching for new antibiotics, prescribing existing antibiotics in a more prudent and less frequent manner, and using combinations of diverse antibiotics to treat infections. In order to successfully employ the latter strategy, it is necessary to utilize a combination of antibiotics that have very different biochemical modes of action.
Fusidic acid is just such an antibiotic. Having a mode of action different than most antibiotics, fusidic acid is unlikely to have cross-resistance with other antibiotics against microorganisms.
A relatively new antibiotic, fusidic acid was discovered in 1962 by Godtfresden and coworkers [See Godtfresden et al., Lancet 1:928 (1962); and, Verbist, J. Antimicrob. Chemotherapy 25: Supp. B:1 (1990)]. It was isolated from the fermentation broth of the fungus Fusidium coccineum. It is a steroid-like antibiotic belonging to the class of the fusidanes, chemically related to cephalosporin P1 and to helvolic acid. Of these fusidanes, however, only fusidic acid has been used clinically with success.
Fusidic acid is most effective against Gram-positive bacteria. In particular, Staphylococcus aureus, S. epidermidis, Clostridium spp. and corynebacteria are highly susceptible [See Verbist supra]. In addition, a few Gram-negative bacteria are susceptible, including Neisseria and Bacteroides spp. However, most Gram negative organisms, including Gram-negative bacilli and fungi, all enterobacteria, Psuedomonas spp., and other non-fermenters are resistant to treatment with fusidic acid. Fusidic acid exhibits moderate efficacy against streptococci, mycobacteria, and Nocardia spp.
Unquestionably, what gives fusidic acid its inherent usefulness in the treatment of microorganisms resistant to other antibiotics is its unique mode of action. Fusidic acid inhibits bacterial protein synthesis by interference with the elongation factor G [See Tanaka et al., Biochem. and Biophys. Res. Commun. 30:278 (1968)]. Such a unique mode of action explains the absence of intrinsic cross-resistance between fusidic acid and any other antibiotics. For example, methicillin-resistant staphylococci are usually susceptible to fusidic acid.
In addition to its usefulness against Gram-positive organisms and bacterial resistance to other antibiotics, there have been recent discoveries related to the use of fusidic acid, which may provide even more clinical benefits.
The use of fusidic acid in treating staphylococcal bone and joint infections has been described [See Coombs, J. Antimicrob. Chemotherapy 25: Supp. B:53 (1990)]. The usefulness of fusidic acid in the treatment of acute osteomyelitis, septic arthritis, chronic osteomyelitis, and other infections encountered in orthopedic surgery merits continued research into the use of fusidic acid for other orthopedic maladies.
Fusidic acid has also recently been shown to be highly effective in treating recurrent bronchopulmonary infections with Staphylococcus aureus suffered by patients having cystic fibrosis [See Jensen et al, J. Antimicrob. Chemotherapy 25: Supp. B:45 (1990)].
Perhaps the most exciting recent discovery is the possible use of fusidic acid in the treatment of AIDS. As described by Barnes in a Science review [238:276 (1994)], fusidic acid was found by researchers in Denmark to have in vitro effectiveness against HIV as well as xe2x80x9cstriking clinical improvementxe2x80x9d in a 58-year-old Danish man stricken with AIDS. These discoveries have led to immediate efforts to determine whether or not fusidic acid will be useful in the treatment of AIDS. So far, the clinical data have been mixed [See Youle et al, J. Acquired Immune Deficiency Syndromes 2:59 (1989); and, Hording et al, Scand. J. Infect. Disease 22:649 (1990)].
With numerous current uses as well as promising future applications, fusidic acid will remain an important pharmaceutical product for the foreseeable future. However, fusidic acid is practically insoluble in water, and the method of choice for oral delivery of the drug is a film coated formulation of sodium fusidate (the sodium salt) or diethanolamine fusidate (the diethanolamine salt). Both derivatives possess significant side-effects including rashes, gastro-intestinal upset, jaundice and other changes in liver function, venospasm, thrombophlebitis, and hemolysis. Clearly, there remains a need for different means of formulation which allows for administration of the agent without inducing serious side-effects.
The present invention relates to novel analogs of fusidic acid. In particular, the present invention relates to novel glycosylated analogs of fusidic acid. The present invention also relates to novel glycosylated analogs of fusidic acid that have different solubility properties than unmodified fusidic acid and that act as chemotherapeutic agents.
A fusidic acid xe2x80x9cderivativexe2x80x9d or xe2x80x9canalogxe2x80x9d of the present invention has the fundamental structure of fusidic acid (see FIG. 1), namely a fused four-ring molecule possessing a steroid-like structure and an alkyl/alkenyl side chain, with either one or more carbohydrate groups attached. The analogs of the present invention have numerous uses. First, they may be successfully employed as standards for analytical techniques (e.g., HPLC) so that new derivatives can be easily identified. Second, the present invention contemplates in vivo use; in accordance with the present invention, a member from the class of novel fusidic acid derivatives is to be delivered as a chemotherapeutic agent, and, in one possible application, to fight anti-microbial infections in the body.
The present invention contemplates derivatives of fusidic acid that have different solubility properties than fusidic acid. These different solubility properties are important because the glycosylated analogs of fusidic acid can be delivered for in vivo use in an admixture with diluent or excipient. It is not intended that the present invention be limited by the nature of the mixture. In one embodiment, the diluent or excipient is propylene glycol. Propylene glycol is miscible in water and a number of organic solvents. Propylene glycol is often used as a substitute for ethylene glycol or glycerol. It can be used as a solvent for oral and injectable drugs and is employed in ointments. [See Goodman and Gilman, The Pharmacological Basis of Therapeutics 9477; and, U.S.P. N.F. 1247]. Dextrose dissolved in an aqueous solution is another diluent or excipient contemplated for this purpose. The aqueous solution used with dextrose can be a buffer or other aqueous solution. For the different diluents or excipient, water or aqueous solutions can be used to dilute the diluent or excipient.
In one embodiment, the present invention contemplates a fusidic acid derivative modified at the C-3 position by chemical, enzymatic, or biological means, such that it contains a carbohydrate unit (See, e.g., FIGS. 2, 3, 10, and 11). In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-24 and C-25 positions by chemical, enzymatic, or biological means, such that the double bonds present at those positions in unmodified fusidic acid are both reduced to single bonds. The later modifications can be in combination with other modifications, such as those outlined in the first embodiment. In another embodiment, the present invention contemplates a glycosylated analog of fusidic acid modified by chemical, enzymatic, or biological means such that (i) the double bonds at the C-2 and C-3 positions of the saccharide unit bound directly to fusidic acid are both reduced to single bonds, and (ii) the double bonds at the C-24 and C-25 positions of the aglycon are both reduced to single bonds.
In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-24 position by chemical, enzymatic, or biological means, such that it has a hydroxyl group. In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-24 position by chemical, enzymatic, or biological means so that the hydroxyl group introduced at the C-24 position has a carbohydrate unit. In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-24 and C-3 positions by chemical, enzymatic, or biological means such that both positions contain carbohydrate units.
In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-25 position by chemical, enzymatic, or biological means, such that it has a hydroxyl group. In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-25 position by chemical, enzymatic, or biological means so that the hydroxyl group introduced at the C-25 position has a carbohydrate unit. In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-25 and C-3 positions by chemical, enzymatic, or biological means such that both positions contain carbohydrate units.
In another embodiment, the present invention contemplates a fusidic acid derivative modified at the C-11 position by chemical, enzymatic, or biological means, such that it contains a carbonyl group as opposed to the hydroxyl group present at that position in unmodified fusidic acid. In another embodiment, the present invention contemplates a fusidic acid derivative modified by chemical, enzymatic, or biological means, such that it contains one or more carbohydrate units having terminal hydroxyl groups which have been deprotected as opposed to having terminal hydroxyl groups which are protected. In yet another embodiment, the present invention contemplates a fusidic acid derivative modified by chemical, enzymatic, or biological means, such that the C-2 and C-3 positions of any saccharide units bound directly to a hydroxyl group of the aglycon are both reduced to single bonds.
For all these embodiments, the present invention contemplates having glycosylated analogs of fusidic acid or glycosylated analogs of modified forms of fusidic acid with either an xcex1- or xcex2-linkage between the oxygen atom of the hydroxyl group of the aglycon and the C-1 position of the saccharide unit bound to fusidic acid. These two types of analogs are known as the xcex1-anomer and the xcex2-anomer of the glycosylated analog.
The contemplated derivations may be prepared in a number of different fashions, and the present invention contemplates many different possible combinations of these derivations giving rise to different fusidic acid analogs.
The carbohydrate unit or units attached to fusidic acid in some of the aforementioned embodiments are exemplified but not limited to 2,3-desoxy-2,3-dehydroglucose, 2,3-desoxy-2,3-dehydroglucose diacetate, glucoside, glucoside tetraacetate, mannoside, mannoside tetraacetate, galactoside, galactoside tetraacetate, alloside, alloside tetraacetate, guloside, guloside tetraacetate, idoside, idoside tetraacetate, taloside, taloside tetraacetate, rhamnoside, rhamnoside triacetate, maltoside, maltoside heptaacetate, 2,3-desoxy-2,3-dehydromaltoside, 2,3-desoxy-2,3-dehydromaltoside pentaacetate, 2,3-desoxymaltoside, lactoside, lactoside tetraacetate, 2,3-desoxy-2,3-dehydrolactoside, 2,3-desoxy-2,3-dehydrolactoside pentaacetate, 2,3-desoxylactoside, glucouronate, N-acetylglucosamine. In one embodiment, the present invention contemplates the use of carbohydrate unit or units having five-membered rings, known as furanoses. In one embodiment, the present invention contemplates the use of carbohydrate unit or units having six-membered rings, known as pyranoses. Combinations of furanoses and pyranoses are also contemplated.
In one embodiment, an analog of the present invention is a glycosylated analog of the fusidic acid molecule of FIG. 1 that has different solubility properties than fusidic acid itself.
In one embodiment, an analog of the present invention is a glycosylated analog wherein fusidic acid is glycosylated at the C-3 position. An example of an analog of the present invention is fusidic acid 3-(4,6-di-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside) [FSA-G-1] (See FIG. 2). Another example of an analog of the present invention is fusidic acid 3-(2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside) [FSA-G-2] (See FIG. 3). Another example of an analog of the present invention is fusidic acid 3-[4-O-(2,3,4,6-tetra-O-acetyl-xcex1-D-glucopyranosyl)-6-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-M-1] (See FIG. 10). Another example of an analog of the present invention is fusidic acid 3-[4-O-(2,3,4,6-tetra-O-acetyl-xcex2-D-galactopyranosyl)-6-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-L-1] (See FIG. 11). Another example of an analog of the present invention is fusidic acid 3-(4,6-bis-O-(chloroacetyl)-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside) [FSA-ClAc-G-1] (See FIG. 12). Another example of an analog of the present invention is fusidic acid 3-[4-O-(2,3,4,6-tetra-O-(chloroacetyl)-xcex1-D-glucopyranosyl)-6-O-(chloroacetyl)-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-ClAc-M-1] (See FIG. 13). Another example of an analog of the present invention is fusidic acid 3-[4-O-(2,3,4,6-tetra-O-(chloroacetyl)-xcex2-D-galactopyranosyl)-6-O-(chloroacetyl)-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-ClAc-L-1] (See FIG. 14). Another example of an analog of the present invention is fusidic acid 3-[4-O-(xcex1-D-glucopyranosyl)-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-M-2] (See FIG. 15). Another example of an analog of the present invention is fusidic acid 3-[4-O-(xcex2-D-galactopyranosyl)-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside] [FSA-L-2] (See FIG. 16).
In one embodiment, an analog of the present invention is a glycosylated analog wherein the secondary hydroxyl group at position C-11 is oxidized to a carbonyl group. An example of an analog of the present invention is 11-dehydrofusidic acid 3-(4,6-di-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside) [FSA-G-3] (See FIG. 4). Another example of an analog of the present invention is 11-dehydrofusidic acid 3-(2,3-dideoxy-xcex1-D-erythro-hex-2-enopyranoside) [FSA-G-4] (See FIG. 5).
In one embodiment, an analog of the present invention is a glycosylated analog wherein fusidic acid is fully reduced at the C-24 and C-25 double bond positions and the C-2 and C-3 positions of the saccharide unit directly bound to the aglycon. An example of an analog of the present invention is 24,25-dihydrofusidic acid 3-(4,6-di-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hexanopyranoside) [FSA-G-5] (See FIG. 6). Another example of an analog of the present invention is 24,25-dihydrofusidic acid 3-(2,3-dideoxy-xcex1-D-erythro-hexanopyranoside) [FSA-G-6] (See FIG. 7). Another example of an analog of the present invention is 11-dehydro-24,25-dihydrofusidic acid 3-(4,6-di-O-acetyl-2,3-dideoxy-xcex1-D-erythro-hexanopyranoside) [FSA-G-7] (See FIG. 8). Another example of an analog of the present invention is 11-dehydro-24,25-dihydrofusidic acid 3-(2,3-dideoxy-xcex1-D-erythro-hexanopyranoside) [FSA-G-8] (See FIG. 9).
In one embodiment, an analog of the present invention is a fusidic acid analog that has protecting groups at positions C-3, C-11, and C-21. (See FIG. 17). In one embodiment, an analog of the present invention is a fusidic acid analog that has C-24. (See FIG. 18). In one embodiment, an analog of the present invention is a fusidic acid analog that has a protecting group at position C-21 and an hydroxyl group at position C-24. (See FIG. 19). In one embodiment, an analog of the present invention is a fusidic acid analog that has protecting groups at positions C-3, C-11, and C-21 and at position C-24 a carbohydrate unit having protecting groups. (See FIG. 20). In one embodiment, an analog of the present invention is a fusidic acid analog that has a carbohydrate unit at position C-24. (See FIG. 21). In one embodiment, an analog of the present invention is a fusidic acid analog that has a protecting group at position C-21 and at each of positions C-3 and C-24 a carbohydrate unit having protecting groups. (See FIG. 22). In one embodiment, an analog of the present invention is a fusidic acid analog that has a carbohydrate unit at each of positions C-3 and C-24. (See FIG. 23). In one embodiment, an analog of the present invention is a fusidic acid analog that has protecting groups at positions C-3, C-11, and C-21 and an hydroxyl group at position C-25. (See FIG. 24). In one embodiment, an analog of the present invention is a fusidic acid analog that has a protecting group at position C-21 and an hydroxyl group at position C-25. (See FIG. 25). In one embodiment, an analog of the present invention is a fusidic acid analog that has protecting groups at positions C-3, C-11, and C-21 and at position C-25 a carbohydrate unit having protecting groups. (See FIG. 26). In one embodiment, an analog of the present invention is a fusidic acid analog that has a carbohydrate unit at position C-25. (See FIG. 27). In one embodiment, an analog of the present invention is a fusidic acid analog that has a protecting group at position C-21 and at each of positions C-3 and C-25 a carbohydrate unit having protecting groups. (See FIG. 28). In one embodiment, an analog of the present invention is a fusidic acid analog that has a carbohydrate unit at each of positions C-3 and C-25. (See FIG. 29).
In one embodiment, an analog of the present invention is synthesized by a) providing in any order: i) unmodified fusidic acid, ii) a derivatizing reagent, and iii) a catalyst; b) reacting in any order: i) said unmodified fusidic acid, ii) said derivatizing reagent, and iii) said catalyst, under conditions such that a glycosylated analog of the fusidic acid molecule of FIG. 1 is formed having different solubility properties than unmodified fusidic acid itself.
In another embodiment, the derivatizing agents are those reagents which provide the substituents added to fusidic acid or a modified form of fusidic acid. In one embodiment, the derivatizing agent is a carbohydrate glycal. In one embodiment, the carbohydrate glycal is either glucose-derived glycal (glucal), lactose-derived glycal (lactal), or maltose-derived glycal (maltal). A catalyst, in general, is a substance which increases the rate of a chemical reaction. In one embodiment, the catalyst is BF3.etherate. In one embodiment, the catalyst is a molecular diatomic halogen. In one embodiment, the molecular diatomic halogen is molecular diatomic iodine.
In one embodiment, the carbohydrate glycal is a disaccharide glycal, for example maltose glycal (maltal), and is synthesized by a) providing in any order: i) unmodified disaccharide, ii) a protecting reagent, iii) a derivatizing reagent, and iv) a reducing agent; b) reacting in any order: i) unmodified disaccharide and ii) a protecting reagent to form a protected disaccharide; c) reacting in any order: i) the protected disaccharide of step (b) and ii) a derivatizing reagent to form a derivatized protected disaccharide; d) reacting in any order: i) the derivatized protected disaccharide of step (c) and ii) an reducing agent to form a disaccharide glycal. In one embodiment, the unmodified disaccharide is maltose. In one embodiment, the unmodified disaccharide is lactose. In one embodiment, the protecting reagent is an esterifying reagent, for example acetic anhydride. In one embodiment, the derivatizing reagent is a halogenating reagent that introduces a halogen atom at the anomeric carbon atom of the carbohydrate, for example hydrobromic acid. In one embodiment, the reducing agent is Zn/CuSO4.
In one embodiment, the carbohydrate glycal is an activated carbohydrate glycal. Activated glycals are those glycals which have a sufficient reactivity to readily react with fusidic acid or a modified fusidic acid to form glycosylated analogs of fusidic acid or of modified fusidic acid. Activated glycals, by definition, are not the parent glycal themselves. Activated glycals are synthesized by a) providing in any order: i) the glycal, ii) an activating reagent, and iii) a catalyst; b) reacting in any order: i) the glycal, ii) a protecting reagent, and iii) a catalyst, under conditions such that an activated carbohydrate glycal is formed. In one embodiment, the carbohydrate glycal is maltal. Activating reagents are those reagents that convert glycals into activated glyeals. In one embodiment, the activating reagent is a carboxylic acid, for example, o-anisic acid. In one embodiment, the catalyst is a molecular diatomic halogen. In one embodiment, the molecular diatomic halogen is molecular diatomic iodine.
In one embodiment, an analog of the present invention is synthesized by a) providing in any order: i) a modified fusidic acid, ii) a derivatizing reagent, and iii) a catalyst; b) reacting in any order: i) said modified fusidic acid, ii) said derivatizing reagent, and iii) said catalyst, under conditions such that a glycosylated analog of a modified form of the fusidic acid molecule of FIG. 1 is formed having different solubility properties than unmodified fusidic acid.
A modified form of fusidic acid is fusidic acid that has been modified by chemical, enzymatic, or biological means so that the modified fusidic acid may still form a glycosylated analog in the aforementioned reaction. In one embodiment, a modified form of fusidic acid is fusidic acid wherein the C-24 and C-25 positions having double bonds have been reduced to single bonds. In another embodiment, the modified form of fusidic acid is fusidic acid wherein the hydroxyl group at C-11 has been oxidized to a carbonyl group. In another embodiment, the modified form of fusidic acid is fusidic acid wherein a hydroxyl group has been introduced at the C-24 position. For this modified form of fusidic acid, the carbon at C-24 may be either one of the two epimers, R or S. This modified form of fusidic acid may consist either of an approximately equal mixture of the two optical isomers at the C-24 position or an excess of one optical isomer over the other. In another embodiment, the modified form of fusidic acid is fusidic acid wherein a hydroxyl group has been introduced at the C-25 position.
For each of these modified forms of fusidic acid, the molecule may have other modifications termed xe2x80x9cprotecting groupsxe2x80x9d which prevent any functional groups of the modified form of fusidic acid from interfering with the glycosylation reaction. It may be the case that depending on whether the modified form of fusidic acid has certain protecting groups, the modified form of fusidic acid may react with the derivatizing reagent to give a glycosylated analog of a modified form of the fusidic acid molecule having more than one carbohydrate unit bound directly through a hydroxyl group to the aglycon. In one embodiment, the protecting group is an acyl. In one embodiment the acyl is monochloroacetyl. In another embodiment, the protecting group is a methyl group.
In one embodiment, an analog of the present invention is synthesized by a) providing in any order: i) a glycosylated analog of fusidic acid having one or more protecting groups and ii) a deprotection agent; b) reacting in any order: i) said glycosylated analog of fusidic acid having one or more protecting groups and ii) the deprotecting reagent to form a glycosylated analog of fusidic acid having fewer protecting groups.
Protecting groups are those groups which prevent undesirable reactions involving unprotected functional groups. In one embodiment, protecting groups protect the terminal hydroxyl groups of a carbohydrate unit. In one embodiment, the protecting group is an acyl. In one embodiment, the acyl is acetate. In one embodiment, the acyl is monochloroacetyl. In one embodiment, the acyl is methoxyacetyl. The prefix xe2x80x9cperxe2x80x9d indicates that all hydroxyl groups in a particular carbohydrate unit are protected by the designated functionality. For example, a xe2x80x9cper-(CIOAc)-glycalxe2x80x9d will have monochloroacetyl groups bound to each hydroxyl group of the carbohydrate unit. Deprotection reagents remove protecting groups. For example, in one embodiment, reaction of a glycosylated analog of fusidic acid with carbohydrate unit or units having protecting acyl groups with a deprotection reagent gives a glycosylated analog of fusidic acid having no protecting groups, i.e., the carbohydrate unit or units have deprotected, free hydroxyl groups. This reaction is commonly known as a hydrolysis reaction. In one embodiment, the deprotecting reagent is a chemical reagent which has properties of a nucleophile. In one embodiment, the deprotecting reagent is Ba(OH)2. In one embodiment, the deprotecting reagent is NaHCO3. In one embodiment, the deprotecting reagent is KHCO3.
In one embodiment, an analog of the present invention is synthesized by a) providing in any order: i) a glycosylated analog of a modified form of fusidic acid having one or more protecting groups and ii) a deprotection agent; b) reacting in any order: i) a glycosylated analog of a modified form of fusidic acid having one or more protecting groups and ii) the deprotecting reagent to form a glycosylated analog of a modified form of fusidic acid having fewer protecting groups.
As for the unmodified fusidic acid, protecting groups can protect the terminal hydroxyl groups of the carbohydrate unit or units. In one embodiment, protecting groups protect functional groups of the aglycon. In one embodiment, the protecting group is an acyl. In one embodiment, the acyl is monochloroacetyl. In one embodiment, the protecting group is methyl. In one embodiment, the deprotecting reagent is Ba(OH)2. In one embodiment, the deprotecting reagent is NaHCO3. In one embodiment, the deprotecting reagent is KHCO3.